Abstract 3404: Detection of circulating tumor DNA using CRISPR-Cas13a in non-small cell lung cancer
Authors: Edwin H. Yau, Bojidar Kandar, Catrina Ting, Sai Yendamuri, Mary Reid
Journal: Cancer Research (2022)

Abstract Background: Lung cancer remains the leading cause of cancer-related death and despite advances in low-dose CT screening, most lung cancer patients still present with advanced disease. The development of assays that are sensitive, specific, economical, and non-invasive may lead to the development of further effective screening strategies. Non-invasive liquid biopsy by detection in plasma of circulating tumor DNA (ctDNA) carrying somatic lung cancer mutations is now routinely used for identifying targeted therapy options in advanced non-small cell lung cancer (NSCLC) patients. Recent adaptation of bacterial CRISPR-Cas13a systems with promiscuous ribonuclease activity into reporter assays for the sensitive and economical detection of specific nucleic acid sequences makes an attractive system for ctDNA detection without needing more costly next-generation sequencing (NGS) techniques. Methods: We adapted the CRISPR-Cas13a system used in the SHERLOCK detection assay for the detection of common driver mutations (KRAS G12C/V, EGFR Exon 19 deletion, and EGFR L858R) in NSCLC and tested the assay on a retrospective cohort of 52 NSCLC patients with known mutations (based on tissue NGS) and with plasma obtained prior to or at the time of any surgical resection and a prospective cohort of 71 patients who underwent lung resection with plasma collected at the time of surgical resection. Results: We designed several CRISPR guide RNAs targeting common EGFR and KRAS oncogenic mutations in NSCLC and were able to generate sensitive CRISPR-Cas13a detection assays against KRAS G12C and G12V, EGFR L858R, and EGFR E746_A750 exon 19 deletion mutations that could identify allele fractions below 1% in reference standards. We then tested plasma of a 52 patient (33 advanced stage IIIB and IV, 19 resectable stage I-IIIA) retrospective cohort with known KRAS and EGFR mutations and matching non-cancer controls using the CRISPR-Cas13a assay as well as with NGS panels (ArcherDx). Using NGS, we were able to confirm ctDNA in 15 samples which we were also able to detect using the CRISPR-Cas13a assay with similar allele frequencies. The CRISPR-Cas13a assay was able to identify ctDNA in 42 out of the 52 retrospective NSCLC samples including 13 out of 19 samples from resectable-stage patients, although the detection rate decreased with earlier stage I and II patients who have a much lower ctDNA level in plasma. We then prospectively collected a cohort of 71 subjects who underwent surgical resection with 38 subjects with lung adenocarcinoma (mostly stage I and II), 11 subjects with benign nodules, and 22 subjects with a different histology malignancy. Our CRISPR-Cas13a assay was able to detect 9 out of 38 lung adenocarcinomas with no false positives in subjects with benign nodules. Conclusion: We adapted a CRISPR-Cas13a assay for the detection of ctDNA in NSCLC and evaluated this assay on plasma from NSCLC subjects. Citation Format: Edwin H. Yau, Bojidar Kandar, Catrina Ting, Sai Yendamuri, Mary Reid. Detection of circulating tumor DNA using CRISPR-Cas13a in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3404.